This invention is in the fields of microfluidics and sensitive detection of analytes. This invention relates to methods and compositions providing rapid, sensitive and specific detection of molecules that are capable of forming strongly associated complexes with a binding molecule-enzyme conjugate.
Although this invention can be applied to both natural and unnatural (man-made and man-designed) products, it is especially useful for the detection of biomolecules. The detection of trace levels of biomolecules is of compelling importance for both scientific research and commercial reasons. The presence and concentration of a biomolecule may give important information with respect to the operation of biochemical pathways. From a practical standpoint, this type of information may have important implications with respect to the detection of certain conditions existing in the body (e.g., changes in hormone levels associated with pregnancy, or with the onset of a variety of metabolic disorders), as well as in the detection of diseases (e.g., by direct detection of a disease agent, or by detection of the presence of the body's response to the presence of a disease agent).
Depending on the particular application, different features may be of greater or lesser importance in a detection method (an assay) for a substance. At a gross level, assays may be divided into those that simply show the presence of a target compound (an analyte), and those that are capable of indicating the concentration of the analyte. Assays of the former type may be sufficient in many cases, one example being that of pregnancy tests: the presence of certain hormones are sufficient to establish that a person is pregnant, and varying concentrations (beyond a certain threshold) will not indicate a state of “more” or “less” pregnant. On the other hand, the ability to accurately quantify the amount of an analyte is critical for many applications in both research and diagnostics. For example, certain hormones (e.g., the thyroid hormones thyroxine and triiodothyronine) are always expected to be present in the human body at some level, but concentrations above or below “standard” levels may indicate some adverse condition (e.g., hypo- or hyperthyroidism). A feature of general desirability in all assays is a high level of sensitivity; the ability to detect a compound (quantitatively or not) at a low concentration. Even when the expected concentration range of a target analyte is much higher than the limit of detection, a more highly sensitive assay will generally be preferred, since it will usually be the case that the reliability of the concentrations determined will be greater than for a less sensitive assay. And, in research studies, a highly sensitive assay may allow data to be obtained over a period of time, making it possible to determine something about the rates of biochemical events.
A feature of assays that is gaining increasing importance as science progresses is the speed of the assay. In a research setting, an assay that is both sensitive and rapidly carried out may make it possible to examine and understand the rates of biochemical processes at a much higher level of detail and understanding. In a clinical setting, assays that are both sensitive and rapid are desirable because they may allow timely diagnosis of rapidly progressing diseases, or make possible so-called “point of care” diagnosis, in which a patient can receive an answer about his/her condition while still at a doctor's office. Of course, other desirable assay features include ease of use and low cost. From a financial standpoint, assays that are capable of determining the presence or concentrations of multiple compounds simultaneously are desirable, since they speed diagnosis for a multitude of potential disease markers.
A wide variety of analytical methods have been developed to meet the needs for the detection of biomolecules. These methods vary in their speed, sensitivity and suitability for use with complex biological samples. In most cases the principle deciding factor in the choice of an assay method is sensitivity. For this reason, two classes of assays have risen to particular prominence in biochemical and clinical applications: methods based on the polymerase chain reaction (PCR), and methods based on enzyme linked immunosorbant assays (ELISA). Both of these methods share a number of common features, the most prominent being that they both involve amplification of a signal by processes that result in an increase in number of detectable species over time. In a PCR assay, a target region of a DNA or RNA molecule is recognized by a complimentary probe molecule, and the sequences are replicated. The resulting copies are then replicated again to give new copies, which are in turn replicated, etc. to give a geometric increase in the number of daughter copies. A variety of strategies have been employed to allow for the specific detection of these daughter copies, but the principle strength of the method lies with the fact that the presence of the original analyte can be inferred (i.e., it is not being directly measured) on the basis of the presence the detectable signal resulting from a given number of amplification (replication) cycles. Although PCR methods appear to be unsurpassed in their sensitivity for the detection of nucleic acid analytes, these methods cannot be applied to non-nucleic acid targets. For the vast array of non-nucleic acid targets, it is the other signal amplification method, ELISA, that is of great use.
A different strategy for signal amplification is used in the ELISA method. There are many variants of this method, but the general theme can be illustrated by what has been termed a “sandwich” ELISA assay. In this variant of the method, an antibody is attached by some means to a surface. When exposed to a sample containing the antigen of the antibody (the analyte), it binds with a high association constant to the surface bound antibody to give the binary complex {surface-antibody}-antigen. After washing away excess sample, the system is exposed to an antibody-enzyme conjugate, wherein the antibody can also bind strongly to the antigen, thereby giving a {surface-antibody}-antibody-{antibody-enzyme} ternary complex. After again washing away unbound materials, the ternary complex is exposed to a solution having a substrate for the enzyme of the antibody-enzyme conjugate. The enzyme and substrate for these processes are chosen so that the substrate is rapidly converted to a detectable reaction product. Since a single enzyme is capable of catalyzing hundreds or thousands of such transformations per minute, signal associated with the presence of the enzyme is amplified accordingly. The amount of signal produced in a unit of time can be used to infer the presence of a certain amount of enzyme, and since (in a perfect world) the amount of enzyme is directly proportional to the amount of analyte/antigen, the amount of analyte can be further inferred. Many variants of this general method exist with respect to the enzyme and corresponding signal producing enzyme catalyzed reactions, as well as in the nature of the complexes formed. However, the methods can be summed up in a general way as involving associating an analyte in some way with a surface, followed by association of an enzyme to this analyte through one or more intervening molecules and/or complexes such that there is an enzyme for each molecule of analyte. Formation of this/these complexes is followed by an enzyme catalyzed reaction that produces some reporter molecule (a detectable ELISA product) that provides a signal that can be detected, with the amount of signal being produced proportional to both the time the reaction is allowed to proceed and the number of enzyme (and, by inference, analyte) molecules present.
The fact that essentially any molecule capable of being bound by one (and preferably in some cases, two) antibody(ies) can be detected with a high degree of sensitivity has led to the development of ELISA methods as the most important class of bioanalytical techniques in both research and clinical settings. Nevertheless, there are problems with the methods—or, at least, areas in which the assay could be dramatically improved to a level that would allow applications of the method that are currently impossible to implement. Two closely related aspects of ELISA methods that, if improved, would greatly increase the attractiveness of the methods, are speed and sensitivity. Because ELISA methods rely on a chemical reaction that produces a detectable product, assay speed and sensitivity are integrally related: if an assay is run for a short time, there will likely be relatively little detectable product that has formed, and as a result the limit of detection (LOD) for that time period will not be low. If one needs greater sensitivity, this can easily be attained by letting the assay run for a longer period of time, thereby providing for greater conversion of the enzyme substrate to detectable product. However, this greater sensitivity will come at the price of a longer assay time. It is not uncommon for ELISA methods to require times ranging from thirty minutes to many hours for the development of sufficient signal for a reliable inference regarding enzyme (and thus, analyte) concentration. This combination of time scale and sensitivity is satisfactory for many applications but not, for example, applications in which the kinetics of moderately rapid biological processes are of interest, or for the rapid sample throughput that would be desirable in point-of-care diagnostic applications. The fact is, it is virtually axiomatic that anything that is capable of increasing the speed or sensitivity of an assay method will be desirable.
The desire to improve the speed and/or sensitivity of ELISA methods has led to a number of innovations in this field. Most improvements of the ELISA method have involved improvements in the signal-to-noise ratio (S/N) in the assay. Improvements in S/N by traditional methods have centered around the construction of new ELISA substrates that will provide reporter products that are more readily detectable by virtue of increased extinction coefficient (for UV-based methods) or fluorescence intensity (for fluorescence based methods). However, the source of sensitivity increase in many of the most dramatically improved versions of ELISA methods lies with a remarkably prosaic source: the decrease, or near elimination of noise (background). In principle, if background noise in an assay could be reduced to nothing, then even a mediocre signal would provide infinite S/N, with an associated infinitely low limit of detection (LOD) for a target analyte. In practice, of course, it is not possible to reduce noise to zero; but it can be brought to very low levels by chemical or instrumental means, or both. Signal enhancement in ELISA by elimination of noise through chemical means can be exemplified by the use of chemiluminescent methods, in which an enzyme substrate is converted to a product that then emits light at a detectable wavelength. Since no other species in the mixture are capable of emitting light (and no incident radiation is applied in the assay) background noise is largely decreased to the point of noise associated with the instrument itself. Signal enhancement in ELISA by elimination of noise through principally instrumental/technological means can be seen in time-resolved fluorescence methods, in which an enzyme substrate is converted to a product that exhibits delayed fluorescence; after an initial burst of radiation, there is a short interval during which no observation takes while most “normal” compounds undergo rapid fluorescent decay. This is followed by an observation period during which only the delayed/long lived fluorescence of the desired reaction product is observed. The absence of even a minor fluorescence background leads to dramatic increases in S/N, and correspondingly large improvements in LOD. Though these methods provide dramatic increases in sensitivity, they do so at a cost. The number of bioluminescent systems that are suitable for generating signal by an enzyme catalyzed reaction are limited and often more costly in terms of synthesis. In many cases, these substrates may have to be stored under special conditions to avoid decomposition. Time-resolved fluorescence requires much more sophisticated instrumentation than that used in simpler methods, leading to analysis systems that are much more costly, and much less portable.